Flavonoids are a diverse family of plant polyphenolic substances. The basic structure of a flavonoid molecule, consists of three phenolic rings referred to as A, B and C rings (FIG. 1). They are derived from a combination of metabolites synthesized from phenylalanine and acetic acid. These compounds include several major subgroups that are found in most higher plants, such as chalcones, flavanones, flavones, flavonols, flavan-3-ols, flavan-4-ols, dihydroflavonols, anthocyanins, proanthocyanidins and condensed tannins. Flavonoids have key roles in signaling between plants and microbes, in male fertility of some species, in defense as antimicrobial agents and in UV protection (Winkel-Shirley, 2001, Plant Physiol., 126(2): 485-493). A general biosynthetic pathway for the flavonoids is shown in FIG. 1.
Flavanones are the common precursors of a wide variety of flavonoids. Their demonstrated anti-oxidant properties and health benefits for a wide array of human pathological conditions have generated a significant research interest in this general area.
Flavones are comprised of two benzene rings linked through a heterocyclic pyrone (Middleton et al., Pharmacol. Rev. 52:673-751, 2000). Flavones, such as chrysin, apigenin and luteolin, exhibit an array of pharmacological properties, including anti-anxiety effects (Viola et al., 1995, Planta Med., 61:213-216; Wolfman et al., Pharmacol. Biochem. 47:1-4, 1994; Wolfman et al., 1995, J. Neurochem. 65:S167), improvement of cardiac function after ischemia (Lebeau, 2001. Bioorg. Med. Chem. Lett. 11:23-27; Rump et al, 1994, Gen. Pharmacol. 25:1137-1142; Schussler et al., 1995, Gen. Pharmacol. 26:1565-1570) and anti-estrogenic effects in breast cancer cell cultures (Miksicek, 1995. P. Soc. Exp. Biol. Med. 208:44-50). Flavones occur only in a relatively small food group that includes parsley, thyme, celery and sweet red pepper (Ross et al, 2002, Annu. Rev. Nutr. 22:19-34).
Flavone and flavanone biosynthesis starts with the conversion of cinnamic acid to p-coumaric acid by a P450 monooxygenase, cinnamate 4-hydroxylase (C4H). p-Coumaric acid is then converted to 4-coumaroyl-CoA by 4-coumaroyl:CoA ligase (4CL). Next, chalcone synthase (CHS) catalyzes a condensation reaction of 4-coumaroyl-CoA with three molecules of malonyl-CoA to form tetrahydroxychalcone. Following this reaction, chalcone isomerase (CHI) performs the stereospecific isomerization reaction of tetrahydroxychalcone to (2S)-flavanone, which is the branch point precursor of many important downstream flavonoids, including flavones. In most cases, a membrane bound cytochromic P450-monooxygenase, flavone synthase II (FSII), catalyzes the biosynthesis of flavones from (2S)-flavanones. However, in certain species of Apiceae, this reaction is performed by the soluble flavone synthase I (FSI) (FIG. 1) (Lukacin et al., 2001, Arch. Biochem. Biophys. 393:177-83; Martens et al., 2001, Phytochemistry 58:43-46).
Among all flavonoid molecules, flavonols are regarded as the most ancient and wide spread (Stafford, 1991). In recent years, their antioxidant activity has attracted much attention due to their potential in the prevention of oxidative stress-related chronic diseases. In that respect, numerous studies have revealed the diverse biological effects of flavonols in such areas as apoptosis induction, antimutagenesis, histamine-release inhibition and angiogenesis inhibition (Formica, et al., 1995; Lambert, et al., 2005; Lamson, et al., 2000).
In the biosynthesis of flavonols, (2S)-flavanones are formed as described above. Natural (2R, 3R)-trans-dihydroflavonols are subsequently formed from (2S)-flavanones by the action of flavanone 3 β-hydroxylase (FHT). Finally flavonol synthase (FLS), a 2-oxoglutarate-dependent dioxygenase, catalyzes the desaturation of dihydroflavonols to flavonols.
Among the natural pigments in plants, anthocyanins are the largest water-soluble group, found in most fruits, flower petals and leaves. The colors range from salmon pink, scarlet and magenta, to violet, purple and blue. These ubiquitous compounds are fascinating in that they can exist in many structural forms, both simple and complex, governed by physiological regulations and chemical modifications which have profound effects on their stabilities and colors. Anthocyanins play important roles such as recruitment of pollinators and seed dispersers, and UV protection. Initial interest in the practical application of these brightly colored anthocyanins has stemmed from their potential as replacements for banned dyes because they have no apparent adverse effects to human health (Brouillard, 1982, Anthocyanins as Food Colors, Academic Press, Inc, New Yor, N.Y.). Recently, however, much attention has been drawn to flavonoid-derived, plant products (including anthocyanins) due to their general antioxidant properties (Kahkonen et al., 2003, J. Agric. Food Chem., 51:628-633; Noda et al., 2000, Toxicology, 148:119-123; Satue-Gracia et al., 1997, J. Agric. Food Chem., 145: 3362-3367) and a consistent association between the consumption of diets rich in fruits and vegetables and a lower risk for chronic diseases, including cancer and cardiovascular disease (Hannum, 2004, Crit. Rev. Food Sci. Nutr., 44:1-17; Middleton et al., 2000, Pharmocolo. Rev., 52:673-751). As a result, anthocyanins are becoming attractive targets for fermentation production from well-characterized microbial hosts, such as Escherichia coli. 
Six major classes of anthocyanidins (the aglycon forms of anthocyanins) exist: pelargonidin, cyanidin, delphinidin, peonidin, malvidin and petunidin. The basic structure of an anthocyanin is a glycosylated form of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium or flavylium salts. Biosynthesis of anthocyanins proceeds via the pathway chalcone→flavanone→dihydroflavonol→anthocyanidin→anthocyanin (FIG. 1). Flavanone is synthesized as described above. Dihydroflavonols are subsequently formed from flavanone by the action of flavanone 3-hydroxylase (FHT). In the next step, dihydroflavonol 4-reductase (DFR) reduces the colorless dihydroflavonols, either dihydrokaempferol (DHK), dihydroquercetin (DHQ) or dihydromyricetin (DHM), to their respective 3,4-cis-leucoanthocyanidins in an NADPH-dependent reaction. The three substrates of DFR are very similar in structure, differing only in the number of additional hydroxyl groups on the β phenyl ring, which are not subject to this enzymatic reaction. DFRs from many plant species (but not all) investigated so far can utilize all three substrates. The colorless, unstable leucoanthocyanidins are the immediate precursors of the first colored metabolite in the biosynthetic pathway, anthocyanidins. This 2-oxoglutarate-dependent reaction is catalyzed by anthocyanidin synthase (ANS). Anthocyanidins are hardly detected in plant tissues, due to their instability. Instead, anthocyanidin 3-glucosides are the first stable colored metabolites from this pathway that are detectable in plants and are derived from anthocyanidins through the action of the enzyme UDP-glucose:flavonoid 3-O-glucosyltransferase (3-GT). The cDNA sequences of a large number of enzymes involved in the anthocyanin biosynthesis pathway from various plant species are now available.
Although attempts have been made to synthesize flavonoids in non-plant systems, a satisfactory system has not been developed. Recently, Hwang et. al. demonstrated the synthesis of plant-specific flavanones for the first time in E. coli, by expressing three genes from heterologous sources that convert phenylalanine to tetrahydroxychalcone. However, the end product (a flavanone) was produced by raising the pH to 9 which spontaneously converted tetrahydroxychalcone to the natural flavanone (2S)-naringenin and its unnatural epimer (2R)-naringenin (hwang et al., 2003, Appl. Environ. Microbiol. 69:2699-2706). Further, so far, no microbial or yeast production has been demonstrated for anthocyanins.
Thus, despite the realization of a need for systems to produce flavonoids in heterologous systems, and the elucidation of cDNA sequences of a large number of enzymes involved in the biosynthetic pathway for flavonoids from various plants, a suitable system and method for the synthesis of variety of flavonoids in microbial systems has not been developed. Therefore, there continues to be a need for the development of methods and systems which can produce usable quantities of flavonoids that can meet the increasing need for those compounds without the need for chemical conversion.